The technique for amplifying a target nucleic acid is one of the exceedingly important techniques in the biotechnology of recent years and is broadly used for the fundamental studies and applications in all manner of fields including biology, medical science, agriculture, forensic medicine, archeology and the like.
1. PCR
Polymerase Chain Reaction (PCR) method is well known as the most typical technique of nucleic acid amplification (e.g., Patent References 1 to 3, Non-patent Reference 1). This method synthesizes a target sequence in vitro, by the action of DNA polymerase activity using two oligonucleotide primers which respectively hybridize with separate DNA chains at both termini of the double-stranded DNA region to be used as the target. Additionally, reverse transcription PCR(RT-PCR) method in which the PCR is combined with reverse transcriptase for the purpose of amplifying a target sequence in RNA is also known (e.g., Non-patent Reference 2). This is a method in which PCR is carried out on a cDNA formed from RNA by the reverse transcription reaction.
In these PCR methods, a specific DNA fragment specified by 5′-ends of the two primers is exponentially accumulated as the amplification product by repeating a reaction consisting of three steps of dissociation (denaturation) of the double-stranded DNA to be used as the template into single-stranded DNA, hybridization (annealing) of the primers to the single-stranded nucleic acid and synthesis (elongation) of template-dependent complementary chain from the primers. Thus, repetition of a total of three steps for adjusting the reaction solution at temperatures which are respectively suited for the above-mentioned three steps (thermal cycle) is required for the PCR method.
One of the useful points of the PCR method is that all parts of the sequence of the target nucleic acid are not necessarily already known, since the amplification reaction proceeds when amplification range of the target nucleic acid is specified by the sequences of two primers (each has a length of about 20 bases or so in general). The fact enables to obtain an unknown nucleic acid sequence by the PCR method from already known limited sequence information. Namely, this is one of the aspects of the reasons why the PCR method has been used for various applications in the broad fields which includes cloning of an unknown gene and preparation of a mutant gene, its use as a preparation method of a nucleic acid with the aim of analyzing an unknown sequence in the subsequent step, and the like.
In the earliest stage of the PCR method, Klenow fragment of Escherichia coli DNA polymerase I was used for the elongation of the annealed primers. Since the denaturation step as a step of the thermal cycle of PCR requires a high temperature which is close to 100° C. and the Klenow fragment is inactivated at said temperature, it was necessary to add fresh enzyme in each cycle. Thus, extremely complex operations were necessary in carrying out the original PCR method. Since the problem was solved by the use of a heat-resistant DNA polymerase in the elongation (e.g., Patent References 4 and 5, Non-patent Reference 3) and the reactions were automated by a temperature cycling device (e.g., Patent Reference 6), the PCR became an easily usable general method.
While complicatedness of the operations of the PCR method was improved in this manner, it left a problem in that the temperature cycling device which controls the reaction temperature and time repeatedly and accurately becomes expensive. Additionally, it is necessary to rise/drop temperature of the reaction liquid over a large number of times during the thermal cycle, and the repetition of time required for the changes in temperature was the cause of prolonging the necessary time until completion of the whole reaction steps. A temperature cycling device which enables high speed temperature changes by minimizing the reaction liquid volume by the use of a glass capillary as the reaction vessel has been developed (e.g., Non-patent Reference 4). Although the use of this device sharply shortened the time required for PCR, the device became further expensive in exchange thereof.
2. SDA
For the purpose of solving such problems, several target nucleic acid amplification methods which can be carried out under an isothermal condition have been developed. As one of them, Strand Displacement Amplification (SDA) is known (e.g., Patent References 7 and 8, Non-patent References 5 and 6). In the method, a 5→3′ exonuclease activity-deficient DNA polymerase (or a strand displacement DNA polymerase) is used as the enzyme which is necessary for the reaction, and a restriction enzyme is also used. During the SDA reaction, the restriction enzyme provides a 3′ end which becomes the starting point of elongation reaction by cutting (nicking) one strand of DNA forming double strand, and the strand displacement DNA polymerase displaces its downstream DNA strand by elongating said 3′ end.
In order to enable the nicking with a restriction enzyme by the SDA method, it is necessary to design the reaction in such a manner that a restriction enzyme recognition sequence to be used is present in a primer-annealed sequence. Furthermore, since a general restriction enzyme digests two strands, in order to allow said enzyme to digest single strand alone, it is necessary that the recognition site is provided as a half-modified (hemi-modified) region where one of the strands has resistance to enzyme digestion. For this purpose, it is necessary to use a large amount of modified dNTP, such as α-S-dNTP in which oxygen atom of the α-position phosphate group is replaced by sulfur atom, as the substrate for DNA synthesis. The necessity of modified dNTP results in the increase in cost of the SDA reaction composition. Additionally, there is a case in which efficiency of the modified dNTP to be incorporated by the DNA polymerase is different from usual dNTP. Since the amplified product of the target formed by the SDA method contains modified nucleotide, use of the amplified product in the subsequent step (e.g., analysis of the presence or absence of digestion and fragment length by digesting the product with a restriction enzyme, gene cloning using the product and the like) is limited.
In the early stage of SDA method, although the reaction proceeded at a constant temperature of from about 37 to 42° C., there is a problem that a background reaction is apt to occur. In order to improve such a problem, a so-called thermophilic SDA method, in which the reaction properly proceeded at a constant temperature of from about 50 to 70° C. by the use of a thermostable enzyme, has been developed (e.g., Patent References 9 and 10). On the other hand, the result narrowed the choices of usable restriction enzymes. One of the advantages of the SDA method is that necessity for an expensive temperature cycling device can be avoided since it proceeds at a single temperature. However, the SDA method is unsuitable for the amplification of long target sequence. Also, when the sequence of a target nucleic acid contains a recognition sequence of the restriction enzyme to be used in SDA in its inner region, amplification of such a target sequence undergoes interference due to the property of principle of SDA. Although the problem can be avoided by changing kind of the restriction enzyme to be used, choices of the usable restriction enzymes are limited. Additionally, when the target nucleic acid contains an unknown sequence, it is difficult to predict generation of this problem.
Some methods have been disclosed for improving disadvantages of the SDA method. For example, the use of restriction enzyme which produces 5′ protruding end (e.g., Patent Reference 11) such as TspRI or the like or the use of nicking endonuclease (e.g., Patent Reference 12) such as N. BstNBI or the like liberates SDA from the limitations which are concerned in the use of modified nucleotide. However, even by such an improved SDA method, all of the above-mentioned problems cannot be avoided as a whole.
3. RCA
As another isothermal target nucleic acid amplification method, a Rolling Circle Amplification (RCA) method which uses a reaction which resembles to the rolling circle type DNA replication found in bacteriophage and the like is conventionally known (e.g., Non-patent Reference 7). In the method, a strand displacement type DNA polymerase elongates a primer on a cyclic template nucleic acid and produces a copy in which complementary chains of the template are continuously ligated. Additionally, high degree amplification is possible by further annealing the primer for said product to elongate its complementary chain. However, it is necessary to provide the RCA method with a cyclic template nucleic acid for the continuous complementary chain synthesis reaction. For the purpose, an additional step, such as ligation using a ligase, is necessary. Additionally, the amplification product of the RCA method becomes a mixture of nucleic acid fragments having different lengths in which a region consisting of the same sequence is repeatedly continued. Accordingly, in order to use the amplification product obtained by the RCA method in the subsequent step, an additional step, such as digestion of the amplification product with a restriction enzyme, becomes necessary in some cases. The necessity for such an additional step is limiting flexibility and convenience of the RCA method.
4. LAMP
As still another isothermal target nucleic acid amplification method, a Loop-mediated Isothermal Amplification (LAMP) method is known (e.g., Patent Reference 13, Non-patent References 8 and 9). In the method, a loop structure is formed by introducing a region, in which the sequence becomes self-complementary, into a terminal region of a target nucleic acid. The 3′ end which becomes the starting point of the elongation reaction is provided by the self-complementary hybridization at the time of the formation of the loop structure or by annealing of a primer to a single-stranded loop region formed by the formation of the loop structure. Said 3′ end is elongated by the action of a strand displacement type DNA polymerase and its downstream DNA chain is displaced.
In order to make the loop structure-mediated DNA synthesis chain reaction possible, it is necessary to provide a template which can form a so-called dumbbell type structure having loop structures on both termini as the starting point structures. To effect this, it is necessary to use appropriately designed four primers which can recognize six regions in the target nucleic acid sequence. Designing of such a primer set sharply increases complexity in comparison with the designing of the primer set for PCR use (a pair of primers which recognize two regions). Primer designing for the LAMP method without the aid of a primer designing support software is an extremely complex operation and easily causes mistakes. The complexity of primer designing of LAMP method is inextricably linked to the high specificity of said amplification reaction for the target.
Another main advantage of the LAMP method is a point that it is a reaction which proceeds at a single temperature without requiring an expensive temperature cycling device and has a markedly high amplification efficiency. However, the LAMP method has a limitation in terms of the amplification of a long target sequence. In general, the length of a template which can become a suitable target of the LAMP method is approximately from about 130 to 300 bp as the region defined by the two inner primers. However, approximately about 80 bases among said template region must have a known sequence in order to design the inner primers. Additionally, in the case of a method which jointly uses a loop primer for the purpose of improving the reaction efficiency (e.g., Non-patent Reference 9), the sequence of approximately about 120 bases among said template region must be already known in order to design the inner primer and loop primer. Accordingly, applications of the LAMP method for amplifying target nucleic acids containing unknown sequences are greatly limited. Additionally, the LAMP method also has a limitation to the amplification of short target sequences. This is because it becomes difficult to form a dumbbell structure suitable for the chain reaction when the length of a sequence to be used as the target is shorter than about 120 bp.
Amplification product of the target nucleic acid obtained by the LAMP method becomes a mixture of nucleic acid fragments having different lengths consisting of a repeating structure having mutually complementary sequences on the same chain. Accordingly, in order to use the amplification product obtained by the LAMP method in the subsequent step, an additional step, such as digestion of the amplification product with a restriction enzyme, is necessary. The necessity for such an additional step is limiting flexibility and convenience of the LAMP method.
5. ICAN
As a further isothermal target nucleic acid amplification method, an Isothermal and Chimeric Primer-initiated Amplification of Nucleic Acids (ICAN) method is known (e.g., Patent Reference 14, Non-patent Reference 10). In the method, a chimeric primer comprising both of a region constituted by a DNA and a region constituted by an RNA is used, and the reaction proceeds by the action of ribonuclease H and a strand displacement type DNA polymerase. In said reaction, the ribonuclease H provides a 3′ end which becomes the starting point of the elongation reaction, by forming a nick through digestion of the RNA chain of the DNA/RNA hybrid part of a double-stranded nucleic acid formed by the annealing of the chimeric primer. On the other hand, the strand displacement type DNA polymerase elongates the thus provided 3′ end and displaces its downstream DNA chain. The ICAN method is also an excellent nucleic acid amplification method from the viewpoint that necessity for an expensive temperature cycling device is excluded. Additionally, as another nucleic acid amplification method which uses a chimeric primer, for example, the methods of Patent References 15 and 16 and the like have also been disclosed.
The amplification method which uses a chimeric primer is confronted by a difficulty that RNA is markedly unstable and apt to be degraded in comparison with DNA. The enzymes which degrade RNA are universally present in organism-derived samples, human perspiration, saliva and skin and various places in the laboratory environments and field environments, and also have such a high heat stability that the activity remains even when they are treated for example at 121° C. using an autoclave. In carrying out its handling and preservation, RNA molecules must be carefully protected from the pollution with the aforementioned degrading enzymes. Additionally, there is a problem that a cost higher than the case of the synthesis of general DNA primers is required for the synthesis of chimeric primers.
6. HDA and RPA
As a still further isothermal target nucleic acid amplification method, a Helicase-dependent Amplification (HAD) method is known (e.g., Patent Reference 17, Non-patent References 11 and 12). In the method, a mechanism of DNA replication in the living body, which is proceeded by a DNA polymerase, a DNA helicase and other accessory protein, is imitated in a test tube. In the HDA method, in order to make annealing of a primer to a template DNA and subsequent elongation by a DNA polymerase possible, a DNA helicase (e.g., UvrD) produces a single-stranded template by separating a double-stranded DNA.
Also, as another isothermal target nucleic acid amplification method, a Recombinase Polymerase Amplification (RPA) method is known (e.g., Patent Reference 18, Non-patent Reference 13). In the method, a recombinase (e.g., uvsX) is allowed to be bound with a primer to form a complex. Said complex (nucleo-protein primer) penetrates into the template double-stranded DNA and enables annealing of the primer to the template. A strand displacement type DNA polymerase elongates said primer and displaces its downstream DNA chain.
The HDA method and RPA method are also excellent nucleic acid amplification methods in terms that an expensive temperature cycling device is not required. However, in the HDA method, it is necessary that cofactors such as ATP, dATP and the like are provided in large amounts during the reaction as energy supplying substances for the helicase activity. Further more, in order to proceed the reaction efficiently in the HDA method, it is necessary in some cases to provide single-stranded DNA binding protein (SSB) such as gp32 and accessory protein such as MutL to the reaction composition for the purpose of supporting the helicase activity (e.g., Non-patent Reference 11). On the other hand, also in the RPA method, a large amount of ATP is required in the reaction liquid as the energy source for the functioning of the recombinase. In addition to this, the presence of SSB such as gp32, a recombinant loading protein such as uvsY and a crowding agent such as polyethylene glycol is essential for the realization of the amplification reaction. Additionally, in the RPA method, it is necessary to allow an ATP regeneration system (e.g., creatine kinase and phosphocreatine) to coexist during the reaction for realizing sufficient amplification efficiency (e.g., Non-patent Reference 13). The necessity for such additional reagents and proteins complicates the reaction composition. It becomes the cause of resulting in a difficulty for optimizing the reaction and of increasing cost of the reaction.
As described in the above, several target nucleic acid amplification methods which can be carried out under an isothermal condition have been devised, and all of them do not require a temperature cycling device and have advantageous points in comparison with the PCR method. Additionally, several methods for amplifying a target nucleic acid under isothermal state have been disclosed other than those exemplified in the above. However, these methods also have respective merits and demerits. Additionally, in some of the nucleic acid amplification methods, due to characteristics of the principle, there are further larger limitations in designing primers in comparison with the designing of the primers for PCR use. When a certain kind of nucleic acid sequence is used as the target, there is a case in which designing of primers for suitably amplifying said sequence is impossible or difficult to attain. Based on such backgrounds, concern has been directed toward the development of a new isothermal target nucleic acid amplification method.    Patent Reference 1: Japanese Patent No. 2093730    Patent Reference 2: Japanese Patent No. 2093731    Patent Reference 3: Japanese Patent No. 2622327    Patent Reference 4: Japanese Patent No. 1814713    Patent Reference 5: Japanese Patent No. 2502041    Patent Reference 6: Japanese Patent No. 2613877    Patent Reference 7: U.S. Pat. No. 5,455,166    Patent Reference 8: U.S. Pat. No. 5,712,124    Patent Reference 9: U.S. Pat. No. 5,648,211    Patent Reference 10: U.S. Pat. No. 5,744,311    Patent Reference 11: International Publication WO99/09211    Patent Reference 12: International Publication WO01/94544    Patent Reference 13: International Publication WO00/28082    Patent Reference 14: International Publication WO00/56877    Patent Reference 15: U.S. Pat. No. 5,916,777    Patent Reference 16: International Publication WO97/04126    Patent Reference 17: International Publication WO04/027025    Patent Reference 18: International Publication WO05/118853    Non-patent Reference 1: Saiki R K, Scharf S, Faloona F, Mullis K B, Horn G T, Erlich H A, Arnheim N: Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 230, p. 1350-1354 (1985)    Non-patent Reference 2: Tomohiro Kinoshita, Kunitada Shimotohno: PCR-ho Ni Yoru RNA No Kaiseki, Tanpakushitsu Kakusan Kohso, 35, p. 2992-3002 (1990)    Non-patent Reference 3: Saiki R K, Gelfand D H, Stoffel S, Scharf S J, Higuchi R, Horn G T, Mullis K B, Erlich H A: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 29, p. 487-491 (1989)    Non-patent Reference 4: Wittwer C T, Ririe K M, Andrew R V, David D A, Gundry R A, Balis U J: The LightCycler, a microvolume multisample fluorimeter with rapid temperature control. Biotechniques, 22, p. 176-181 (1997)    Non-patent Reference 5: Walker G T, Little M C, Nadeau J G, Shank D D: Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. Proc Natl Acad Sci USA, 89, p. 392-396 (1992)    Non-patent Reference 6: Walker G T, Fraiser M S, Schram J L, Little M C, Nadeau J G, Malinowski D P: Strand displacement amplification—an isothermal, in vitro DNA amplification technique. Nucleic Acids Res, 20, p. 1691-1696 (1992)    Non-patent Reference 7: Lizardi P M, Huang X, Zhu Z, Bray-Ward P, Thomas D C, Ward D C: Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet, 19, p. 225-232 (1998)    Non-patent Reference 8: Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T: Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 28, E63 (2000)    Non-patent Reference 9: Nagamine K, Hase T, Notomi T: Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes, 16, p. 223-229 (2002)    Non-patent Reference 10: Masamithsu Shimada, Fumitsugu Hino, Hiroaki Sagawa, Hiroyuki Mukai, Kiyozo Asada, Ikunoshin Kato: To-on idenshi zoshoku-ho (ICAN) ni yoru kekkaku kin kenshutsu shiyaku no kaihatsu, Rinsho Byori, 50, p. 528-532 (2002)    Non-patent Reference 11: Vincent M, Xu Y, Kong H: Helicase-dependent isothermal DNA amplification. EMBO Rep, 5, p. 795-800 (2004)    Non-patent Reference 12: An L, Tang W, Ranalli T A, Kim H J, Wytiaz J, Kong H: Characterization of a thermostable UvrD helicase and its participation in helicase-dependent amplification. J Biol Chem, 280, p. 28952-28958 (2005)    Non-patent Reference 13: Piepenburg O, Williams C H, Stemple D L, Armes N A: DNA detection using recombination proteins. PLoS Biol, 4, e204 (2006)